1. Field of the Invention
The present invention relates to instruments for analyzing a multiplicity of fluorescent dyes using a multiplicity of photodetectors. The present invention is particularly applicable to the field of cytometry, more particularly, flow or scanning cytometry.
2. Description of Related Art
Particle analyzers, such as flow and scanning cytometers, are well known in the art. In these systems, fluorescently labeled particles, such as molecules, analyte-bound beads, or individual cells, are individually analyzed by exposing each particle to an excitation light, typically one or more lasers, and measuring the resulting fluorescence from each of dye labels. Each particle may be labeled with a multiplicity of spectrally distinct fluorescent dyes. Typically, detection is carried out using a multiplicity of photodetectors, one for each distinct dye to be detected. Both flow and scanning cytometers are commercially available from, for example, BD Biosciences (San Jose, Calif.).
Other instruments systems are known to be capable of detecting a multiplicity of fluorescent dyes using a multiplicity of photodetectors. For example, nucleic acid amplification reaction products from multiple target sequences can be detected and distinquished using fluorescently labeled probes, wherein each target-specific probe is bound to a spectrally distinct dye. Typically, an instrument for analyzing nucleic acid amplification products measures the total fluorescence from a reaction mixture, and the frequency of each target species is determined from the measured fluorescence from each dye.
In flow cytometers and other instruments that employ a multiplicity of photodetectors to detect a multiplicity of dyes, the collected light is separated into specific ranges of wavelengths, typically by a system of frequency-dependent filters and dichroic mirrors, such that the light detected by a particular photodetector is limited to a predefined range of wavelengths, referred to as a detection channel. The detection channels and dyes are selected such that the peak of the emission spectrum of each dye is within the frequency range of a different detection channel, i.e., each detection channel detects primarily the emission from a single dye. However, because of the breadth of the emission spectra of fluorescent dyes, typically a dye will fluoresce in more than one detection channels and, thus, measurements of dye fluorescence are not independent. The emission of one dye in detection channels intended for the detection of other dyes is referred to by a number of terms, such as spillover, fluorescence spectral overlap, and crosstalk.
Methods of decreasing the effect of spillover or crosstalk on the dye fluorescence measurements are known in the art. Such methods involve adjustment of the signal measured by each photodetector by an amount calculated to compensate for the contribution from dyes other than the primary dye to be detected. Examples in the field of flow cytometry include Bagwell, C. B.; Adams, E. G. “Fluorescence Spectral Overlap Compensation for any Number of Flow Cytometer Parameters”, Ann. N.Y. Acad. Sci. 677, 167-184 (1993); Roederer, M. et al., “Eight Color, 10-Parameter Flow Cytometry to Elucidate Complex Leukocyte Hetrogeneity”, Cytometry 29, 328-339 (1997); and Bigos et al., 1999, Cytometry 36: 36-45, each incorporated herein by reference. WinList™ (Verity Software House, Topsham, Me.) is a stand-alone software package that allows software compensation on the stored data files produced by a flow cytometer. See also the whitepaper describing the BD FACSDiVa™ Option for the BD FACSVantage SE Flow cytometer (BD Biosciences, San Jose, Calif.; available at www.bdbiosciences.com), incorporated herein by reference.
In a typical flow cytometric analysis, labeled particles suspended in a liquid medium are passed through a narrow channel one at a time past an interrogation region. Particles are labeled with one or more fluorescent dyes to facilitate identification. While passing the interrogation region, labeled particles are exposed to excitation light, typically from one or more lasers, and the resulting particle fluorescence is measured. Typically, the amount of excitation light scattered by the particles also is measured. The amount of scattered light and the intensity of emitted fluorescent light from each of the bound labels provide a characterization of the labeled particles. Flow cytometry provides a rapid means of analyzing a large number of particles and, importantly, provides data on each individual particle, rather than only on the particle population as a whole. However, the detection of low level of light emitted by the dye molecules bound to a single particle typically requires amplification of the detected signal. To detect such low levels of emitted light, current flow cytometers use photodetectors such as photomultiplier tubes (PMT) and avalanche photodiodes (APD) that are capable of amplifying the signal. Such photodetectors are capable of amplifying the signal amplifiction by a factor of 106 or greater. The amplification gain of a PMT or APD can be varied by adjusting an input voltage to the detector, or by adjusting the gain of a downstream amplifier, or both.
Instruments for the detection of labeled nucleic acid amplification products typically measure labeled products at the population level, rather than at the level of individual particles, and the degree of signal amplification required depends on the volume of sample analyzed. Signal amplification, if used, can be achieved using an amplifier in-line with the detector output. As with a PMT or APD, the amplification gain typically is adjustable.
Prior to carrying out a particular assay using a flow cytometer, photodetector signal amplification (gain) and the signal range detected are adjusted based on the brightness/amount of dyes to be detected in order that the sample measurements are within the dynamic range of the detection system. To provide maximum resolution of sample fluorescence level, it is desirable that the photodetector gain and the detected signal range are set such that the expected range of sample fluorescence spans a significant portion of the detectable range. As the expected range of sample fluorescence is sample-specific, these instrument parameters must be determined and set prior to analyzing each kind of sample. In addition, these parameters are specific to the instrument, as individual instruments will differ in their performance.
Photodetector gain and the detected signal range typically are set in a flow cytometer by analyzing samples of standards that are representative of the unknown sample to be analyzed subsequently. For example, before analyzing a cell-containing sample, a sample of beads or cells dyed with an amount of dye representative of the expected brightness of a brightly-dyed cell is used to set the upper end of the detection range, or a sample of unlabeled beads or cells that fluoresce at a level of an unlabeled sample cell are used to set the lower end of the detection range. This determination of appropriate settings typically is carried out each day, even if the same type of analysis is to be carried out each day, in part because of day-to-day variation in instrument and photodetector performance.
Because the levels of photodetector gain in each of the multiple photodetectors affects the measurement of light in each channel, the amount of spillover fluorescence measured is dependent on the photodetector gains. Using current flow cytometers, the relative amounts of spillover fluorescence from the dyes, used to determine compensation, are experimentally determined after the photodetector gain settings have been chosen. Any change to the instrument's photodetector gain settings after the initial setup renders the measurements of spillover and, hence, the compensation, no longer applicable to current instrument settings. The relative amounts of spillover fluorescence from the dyes must be redetermined experimentally using the current instrument settings, and the compensation is redetermined from the experimental results.